This revised competitive renewal proposal focuses on the mechanisms by which febrile seizures (FS), the most prevalent seizure type in infants and children, may lead to Epilepsy. The applicant has characterized an immature rat model of prolonged FS, those associated with subsequent development of limbic, 'temporal lobe' epilepsy (TLE) in human studies. During the current award period, it was discovered that experimental prolonged FS lead to limbic epilepsy in approximately 35% of animals, establishing the paradigm as a valid model of developmental epileptogenesis. In addition, these seizures induced changes in the expression and co- assembly of specific ion channels, the hyperpolarization-activated, cyclic nucleotide gated (HCN) channels, that promoted hippocampal hyper-excitability. Remarkably, changes in the expression and in the relative abundance of the same members of the HCN channel family were found also in human hippocampus from individuals with TLE and a history of early-life seizures, confirming the relevance of molecular changes in these channels to clinical epileptogenesis. The current proposal aims to address important gaps in our understanding of the epileptogenic process bridging prolonged 'FS' and limbic epilepsy: 1) First, in vitro systems will be used to determine how these developmental seizures lead to down-regulation of the HCN1 isoform expression and increased expression of HCN2, by testing hypotheses about seizure-evoked, calcium-mediated cellular cascades influencing coordinate HCN isoform expression; 2) To determine how seizures evoke re-arrangements of HCN channel molecules into heteromeric channels that promote hyper-excitability: the role of post-translational glycosylation of the channels in the expression of heteromeric channels will be studied in vitro; 3) Finally, in vivo experiments will investigate the parameters of the inciting FS that govern epileptogenesis, aiming to generate a more powerful model, where seizures elicit epilepsy in the majority of rats. Public health relevance (lay language): Epilepsy is the most common chronic brain disorder in young individuals. Temporal lobe epilepsy is the most refractory epilepsy, and is associated with early-life prolonged febrile seizures. Understanding the steps and the mechanisms by which febrile seizures convert a 'normal' hippocampus to an epileptic one is a critical first step in intercepting the epilepsy-generating process resulting in temporal lobe epilepsy.